justification of the Reproduction and genetics work planned in diversify

Reproductive control has been identified as the basis of sustainable aquaculture production. Inadequate reproductive control limits the production of market-sized fish, which may also depend on the unsustainable capture of wild organisms. For example, Pangasius spp catfish did not complete maturation in captivity and the development of hormone induced spawning (Cacot et al., 2002) enabled unlimited production of eggs, larvae and juveniles removing this bottleneck to provide the conditions for Vietnam to increase Pangasius spp aquaculture production from 40,000 tonnes in 1997 to 376,000 tonnes in 2005 (FAO, 2012). A reproductive bottleneck to the production of a species new to aquaculture is principally caused by two types of reproductive dysfunctions, related to failure of final oocyte maturation (FOM) and ovulation, and/or failure to spawn after ovulation (Mylonas et al., 2010a). The exact cause of these dysfunctions is unclear, but is probably related to inadequate culture environment (physical and/or social) and nutrition, which do not provide the optimal conditions that enable maturation to be completed (Mylonas et al., 2010a; Duncan et al., 2013). The lack of maturation is controlled by the endocrine brain-pituitary-gonad axis and Zohar et al. (1995) demonstrated that gilthead sea bream arrested in late vitellogenesis produced gonadotropins in the pituitary, but gonadotropin releasing hormone (GnRH) was not produced by the hypothalamus to release the gonadotropins and stimulate oocyte maturation and ovulation. Broodstock nutrition clearly plays a role in the progress of maturation and work within the consortium (Norambuena et al., 2012; 2013) suggested that inadequate dietary arachidonic acid (ARA) and cholesterol affected negatively the production of reproductively important prostaglandins and steroids. DIVERSIFY will study the underlying endocrine and nutritional status of reproductive dysfunctions in some of the proposed species for Aquaculture diversification, which have been demonstrated to present significant reproductive dysfunctions, preventing the reliable production of eggs and, thus, juveniles for grow out.This approach has proven results in other species, such as European sea bass (Mylonas et al., 2003) and Atlantic bluefin tuna (Thunnus thynnus) (Mylonas et al., 2007, 2010b). The work in DIVERSIFY, will advance the state-of-the-art in the understanding of the mechanisms that control reproductive dysfunctions in these new species, and produce reproduction control protocols for the EU aquaculture industry. Despite of the incomplete understanding of the causes of reproductive dysfunctions, solutions, hormone therapies and strip spawning have been developed by consortium members of DIVERSIFY and used successfully by the industry in a wide range of species (reviewed by Mylonas et al., 2010a; Duncan et al., 2013). The hormone therapies used to induce FOM, ovulation and spawning in aquaculture include the application of exogenous gonadotropins (GtH) or agonists of gonadotropin releasing hormone (GnRHa) to fish at the correct stage of maturity. The recent development of recombinant fish GtHs offers a source of exogenous, but homologous hormone for induction therapies that may be targeted to specific receptors, are species specific and without many of the disadvantages of other sources of heterologous GtHs, such as immune reactions and spread of pathogens (Yaron et al., 2009; Rosenfeld et al., 2011; Chauvigné et al., 2012). In the aquaculture industry, egg production from species that fail to spawn after ovulation is based on stripping eggs that are ovulated spontaneously or by hormonal induction (Mylonas et al., 2010a). Strip spawning protocols identify the hormonal therapy to induce ovulation (if required), when ova must be striped to avoid over-ripening and how sufficient fresh, cooled or cryopreserved sperm is obtained (Duncan et al., 2013a). Combinations of these two solutions will be used in DIVERSIFY to solve the reproductive bottlenecks documented in the chosen species. In the greater amberjack, oogenesis often did not proceed in a predictable and reliable way in captivity (Micale et al., 1999¸ Mylonas et al., 2004). Work within the consortium has reported spontaneous spawning in a single female held in captivity in the eastern Atlantic (Jerez et al., 2006) and in the Mediterranean Sea, spawning of good quality eggs from a single female with GnRHa implants (Mylonas et al., 2004). Kozul et al. (2001) induced spawning with heterologous GtH (hCG), but all eggs died in the gastrula stage. Recent comparisons between the nutritional status of wild and cultured amberjack broodstock have indicated how diets could be improved (Rodríguez-Barreto et al., 2012). With collaboration between Reproduction and Genetics, and Nutrition on the development of an appropriate amberjack broodstock diet and the application of the most recent knowledge on spawning induction methods (Mylonas et al., 2010a), DIVERISFY will develop GnRHa-based spawning protocols for the greater amberjack. In excess of 100,000 t of grey mullet (FAO, 2012) are produced from wild juveniles, due to lack of egg availability from cultured broodstocks (Aizen et al. 2005). Spermiating males are rarely observed and in most cases the produced milt is highly viscous and fails to fertilize the eggs (Yashouv, 1969; De Monbrison et al., 1997). Females are often arrested in the early stages of vitellogenesis, or fail to undergo oocyte maturation and ovulation (De Monbrison et al., 1997). In a previous study, DIVERSIFY Partners described dopamine inhibition of GnRH (Aizen et al. 2005) and induced spawning with controlled-release GnRHa implants combined with dopamine antagonist and 17α-methyl-testosterone (Aizan et al 2005). Work also demonstrated that recombinant tuna FSH induced spermiation in grey mullet (Rosenfeld et al., 2011). Building on these advances, DIVERSIFY will establish an optimized hormonal therapy for grey mullet in captivity, leading to mass-scale egg production. Furthermore, DIVERSIFY will evaluate the effects of captivity on first sexual maturity to establish strategies for production of an additional high-value product (mullet roe) with minimal extra risks and/or costs. Mullet roe (known also as "bottarga" or "Karasumi") is a high-priced product in Mediterranean and Asian markets, but fully developed vitellogenic roe (10-20% GSI) in nature occurs first in 3 year-old fish. A reduction of age of maturity by a year will enhance the cost-efficiency of grow out production by the Aquaculture industry. The availability of wreckfish broodstocks is limited throughout the world (Brick & Klippel, 2003; Cergole et al., 2005) and consequentially few reproductive studies have been completed. DIVERSIFY Partners have maintained fish in captivity for some years and obtained important information on reproduction, including gametogenesis (Papandroulakis et al., 2004), strip spawning of an ovulated female (Peleteiro et al., 2011) and GnRHa induced spawning and in vitro fertilization (Fauvel et al., 2008; Papandroulakis et al., 2008). These studies indicate that wreckfish may require induction of FOM and ovulation combined with strip spawning. DIVERSIFY will build on these advances to enhance our knowledge of wreckfish reproduction and broodstock management, by describing gamete characteristics and develop hormone induction and in vitro fertilization protocols. Wild Atlantic halibut broodstock mature in captivity and are strip-spawned in relation to the natural ovulatory rhythms of individual fish (Norberg et al., 1991). Until recently, a bottleneck preventing the expansion of the industry was the slow growth of males. Artificial sex reversal of females (Hendry et al., 2003; Babiak et al., 2012) and breeding of the produced phenotypic males (that are genotypic females) has established all female production in the industry in Norway and North America, and has greatly ameliorated this bottleneck. However, F1/F2 females produced in a hatchery display a high degree of reproductive dysfunction, with irregular spawning cycles, low and unstable fertilisation rates, low survival of gametes, and lower fecundity than wild females. DIVERSIFY will examine controls of fecundity and apply hormone induction technologies to develop solutions to poor F1/F2 Atlantic halibut spawning performance. Control of reproduction of meagre and pikeperch is not considered as a bottleneck. DIVERSIFY Partners have developed spawning induction protocols for meagre (Fernández-Palacios et al., 2009, 2011; Mylonas et al., 2011; Duncan et al., 2012) and pikeperch (Kestemont & Melard, 2000; Zarski et al., 2012). However, the industry identified two genetically related bottlenecks, which are unknown genetic variability in captive broodstocks and unpredictable growth and performance during grow-out. A solution to both of these bottlenecks in other species has been achieved with genetic breeding programs that control rates of inbreeding and improve growth with every generation to form an important part in maintaining the competitiveness of an aquaculture business (Gjedrem, 2012; Duncan et al., 2013a). Microsatellite-based studies on meagre (Haffray et al., 2012) and pikeperch (Saisa et al., 2010; Saliminen et al., 2011) have characterised wild populations. Within the consortium, microsatellites have been identified from the meagre genome (Andree et al., 2010) and used for paternity analysis and to characterise broodstocks (Duncan et al., 2012; 2013b). To date, no studies have been published that genetically characterise captive pikeperch broodstocks or analyze the genetic basis of phenotypic traits such as growth in either species. DIVERSIFY will characterise genetically available cultured broodstocks of both species, and fast/slow growers for meagre. DIVERSIFY will also provide tools that the industry can use to establish genetic breeding programs, improving on and developing new genetic tools for meagre and pikeperch. These include protocols to produce planned family crosses for meagre using paired tank spawning, in vitro fertilization, and characterisation of sperm and sperm management procedures. Protocols for pikeperch planned family crosses have been developed already (Kestemont & Melard, 2000; Zarski et al., 2012).